Direct Target Of miR-21 Research Articles

Anti-miR-21 oligonucleotide enhances chemosensitivity of leukemic HL60 cells to arabinosylcytosine by inducing apoptosis

Drug insensitivity or resistance is a major obstacle for successful treatment of acute myeloid leukemia. MicroRNAs (miRNAs) are small non-coding RNA molecules. Increasing evidence suggests that miRNAs modulate cellular sensitivity to anticancer drugs. We used a specific anti-miR-21 oligonucleotide (AMO-miR-21) to sensitize leukemic HL60 cells to arabinosylcytosine (Ara-C) by down-regulating miR-21. AMO-miR-21 alone effectively inhibited HL60 cell viability as measured by MTT assays and induced apoptosis as evaluated by flow cytometry, whereas AMO-miR-21 in combination with Ara-C enhanced HL60 cells to Ara-C-sensitivity and promoted Ara-C-induced apoptosis. Levels of miR-21 and its target PDCD4, quantified by real-time PCR, showed that expression of miR-21 was significantly decreased after AMO-miR-21 treatment. PDCD4 as a direct target of miR-21 in leukemic HL60 cells was confirmed by the dual-luciferase reporter assay. Our study suggests that AMO-miR-21 significantly sensitizes HL60 cells to Ara-C by inducing apoptosis and these effects of AMO-miR-21 may be partially due to its up-regulation of PDCD4. Therefore, exploiting synergistic effects between AMO-miR-21 and Ara-C might be an effective clinical strategy for leukemia chemotherapy.

MicroRNA-21 is involved in osteosarcoma cell invasion and migration

MicroRNAs are involved in different cancer-related processes. MicroRNA-21 (miR-21), as an oncomiR, is overexpressed in all kinds of tumors and the role of miR-21 in carcinogenesis is elucidated in many cancers gradually. However, the function of miR-21 in osteosarcoma is still unclear. In our study, we found that miR-21 was significantly overexpressed in osteosarcoma tissues. More importantly, we confirmed that knockdown of miR-21 greatly decreased cell invasion and migration of MG-63. Furthermore, we identified that RECK (reversion-inducing-cysteine-rich protein with kazal motifs), a tumor suppressor gene, was a direct target of miR-21. Finally, the expression of RECK protein negatively correlated with the expression of miR-21 in human osteosarcoma tissues, indicating the potential regulation of RECK by miR-21. Our results suggest that miR-21 expression has a key role in regulating cellular processes in osteosarcoma, likely through regulating RECK and may serve as a therapeutic target.

<italic>BMPRII</italic> is a direct target of miR-21

MicroRNAs (miRNAs) are a type of small non-coding RNAs that regulate cognate mRNA expressions at the post-transcriptional stage. Although several miRNAs are known to be involved in various biological processes, including developmental timing, patterning, embryogenesis, differentiation and organogenesis, growth control, and apoptosis, many target genes and the functions of most miRNAs are still unclear. Since there is only a partial complementarity between miRNAs and their targets in animal cells, it is difficult to identify the specific target genes for a given miRNA and elucidate its function. In this study, we confirmed that bone morphogenetic protein receptor II (BMPRII) is a direct target of miR-21, and also showed that the protein level of BMPRII correlates inversely with the amount of miR-21 in PC3 and Lncap cells. These findings suggest that miR-21 may have a potential role in regulating the malignancy and metastatic abilities of prostate cancer cells and in self-renewal of stem cells by regulating the expression of BMPRII.

MicroRNA-21 Is Up-Regulated in Allergic Airway Inflammation and Regulates IL-12p35 Expression

Allergic airway inflammation is characterized by marked in situ changes in gene and protein expression, yet the role of microRNAs (miRNAs), a new family of key mRNA regulatory molecules, in this process has not yet been reported. Using a highly sensitive microarray-based approach, we identified 21 miRNAs with differential expression between doxycycline-induced lung-specific IL-13 transgenic mice (with allergic airway inflammation) and control mice. In particular, we observed overexpression of miR-21 and underexpression of miR-1 in the induced IL-13 transgenic mice compared with control mice. These findings were validated in two independent models of allergen-induced allergic airway inflammation and in IL-4 lung transgenic mice. Although IL-13-induced miR-21 expression was IL-13Ralpha1 dependent, allergen-induced miR-21 expression was mediated mainly independent of IL-13Ralpha1 and STAT6. Notably, predictive algorithms identified potential direct miR-21 targets among IL-13-regulated lung transcripts, such as IL-12p35 mRNA, which was decreased in IL-13 transgenic mice. Introduction of pre-miR-21 dose dependently inhibited cellular expression of a reporter vector harboring the 3'-untranslated region of IL-12p35. Moreover, mutating miR-21 binding sites in IL-12p35 3'-untranslated region abrogated miR-21-mediated repression. In summary, we have identified a miRNA signature in allergic airway inflammation, which includes miR-21 that modulates IL-12, a molecule germane to Th cell polarization.

Involvement of MicroRNAs in Hydrogen Peroxide-mediated Gene Regulation and Cellular Injury Response in Vascular Smooth Muscle Cells

MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate approximately 30% of genes in a cell via degradation or translational inhibition of their target mRNAs. However, the effects of reactive oxygen species (ROS) on miRNA expression and the roles of miRNAs in ROS-mediated gene regulation and biological functions of vascular cells are unclear. Using microarray analysis, we demonstrated that miRNAs are aberrantly expressed in vascular smooth muscle cells (VSMCs) after treatment with hydrogen peroxide (H(2)O(2)). H(2)O(2)-mediated up-regulation of microRNA-21 (miR-21) was further confirmed by quantitative real-time PCR. To determine the potential roles of miRNAs in H(2)O(2)-mediated gene regulation and cellular effects, miR-21 expression was down-regulated by miR-21 inhibitor and up-regulated by pre-miR-21. H(2)O(2)-induced VSMC apoptosis and death were increased by miR-21 inhibitor and decreased by pre-miR-21. Programmed cell death 4(PDCD4) was a direct target of miR-21 that was involved in miR-21-mediated effects on VSMCs. Pre-miR-21-mediated protective effect on VSMC apoptosis and death was blocked via adenovirus-mediated overexpression of PDCD4 without the miR-21 binding site. Moreover, activator protein 1 was a downstream signaling molecule of PDCD4 in miR-21-modulated VSMCs. The results suggest that miRNAs in VSMCs are sensitive to H(2)O(2) stimulation. miRN-21 participates in H(2)O(2)-mediated gene regulation and cellular injury response through PDCD4 and the activator protein 1 pathway. miRNAs might play a role in vascular diseases related to ROS.

Identification of miR‐21 targets in breast cancer cells using a quantitative proteomic approach

MicroRNA (miRNA) play essential roles in biological processes ranging from cellular proliferation to apoptosis. Recently, miRNA have also been implicated in a number of diseases including cancers. However, the targets of most miRNA remain unknown. The majority of reports describing identification of miRNA targets are based on computational approaches or detection of altered mRNA levels despite the fact that most miRNA are thought to regulate their targets primarily at the level of translational inhibition in animals. miR-21 is a miRNA with oncogenic activity that is involved in various cancer-related processes such as invasion and migration. Given the importance of miR-21 in tumorigenesis, we employed a quantitative proteomic strategy to systematically identify potential targets of miR-21. By knocking down the expression of endogenous miR-21 in MCF-7 breast cancer cells, we observed an increase in the abundance of 58 proteins, implying that they could be potential targets of miR-21. Validation of 12 of these candidate targets in luciferase assays showed that 6 of them were likely direct targets of miR-21. Importantly, the mRNA of the majority of the candidate targets tested did not show a concomitant increase in abundance. Overall, our results demonstrate that miR-21 affects the expression of many of its targets through translational inhibition and highlights the utility of proteomic approaches for identifying miRNA targets.

MicroRNA-21 protects against the H2O2-induced injury on cardiac myocytes via its target gene PDCD4

Reactive oxygen species (ROS)-induced cardiac cell injury via expression changes of multiple genes plays a critical role in the pathogenesis of numerous heart diseases. MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate about 30% of the genes in a cell via degradation or translational inhibition of their target mRNAs. Currently, the effects of ROS on miRNA expression and the roles of miRNAs in ROS-mediated injury on cardiac myocytes are uncertain. Using quantitative real-time RT-PCR (qRT-PCR), we demonstrated that microRNA-21 (miR-21) was upregulated in cardiac myocytes after treatment with hydrogen peroxide (H2O2). To determine the potential roles of miRNAs in H2O2-mediated gene regulation and cellular injury, miR-21 expression was downregulated by miR-21 inhibitor and upregulated by pre-miR-21. H2O2-induced cardiac cell death and apoptosis were increased by miR-21 inhibitor and was decreased by pre-miR-21. Programmed cell death 4 (PDCD4) that was regulated by miR-21 and was a direct target of miR-21 in cardiac myocytes. Pre-miR-21-mediated protective effect on cardiac myocyte injury was inhibited in H2O2-treated cardiac cells via adenovirus-mediated overexpression of PDCD4 without miR-21 binding site. Moreover, Activator protein 1 (AP-1) was a downstream signaling molecule of PDCD4 that was involved in miR-21-mediated effect on cardiac myocytes. The results suggest that miR-21 is sensitive to H2O2 stimulation. miR-21 participates in H2O2-mediated gene regulation and functional modulation in cardiac myocytes. miR-21 might play an essential role in heart diseases related to ROS such as cardiac hypertrophy, heart failure, myocardial infarction, and myocardial ischemia/reperfusion injury.

MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level by either degradation or translational repression of a target mRNA. Encoded in the genome of most eukaryotes, miRNAs have been proposed to regulate specifically up to 90% of human genes through a process known as miRNA-guided RNA silencing. For the first time, we sought to test how myocardial ischaemia-reperfusion (IR) changes miR expression. Following 2 and 7 h of IR or sham operation, myocardial tissue was collected and subjected to miRNA expression profiling and quantification using a Bioarray system that screens for human-, mice-, rat-, and Ambi-miR. Data mining and differential analyses resulted in 13 miRs that were up-regulated on day 2, 9 miRs that were up-regulated on day 7, and 6 miRs that were down-regulated on day 7 post-IR. Results randomly selected from expression profiling were validated using real-time PCR. Tissue elements laser-captured from the infarct site showed marked induction of miR-21. In situ hybridization studies using locked nucleic acid miR-21-specific probe identified that IR-inducible miR-21 was specifically localized in the infarct region of the IR heart. Immunohistochemistry data show that cardiac fibroblasts (CFs) are the major cell type in the infarct zone. Studies with isolated CFs demonstrated that phosphatase and tensin homologue (PTEN) is a direct target of miR-21. Modulation of miR-21 regulated expression of matrix metalloprotease-2 (MMP-2) via a PTEN pathway. Finally, we noted a marked decrease in PTEN expression in the infarct zone. This decrease was associated with increased MMP-2 expression in the infarct area. This work constitutes the first report describing changes in miR expression in response to IR in the mouse heart, showing that miR-21 regulates MMP-2 expression in CFs of the infarct zone via a PTEN pathway.

Hypothesis of mechanism of microRNA-directed DNA methylation and its evolutionary descent

Reactive oxygen species (ROS)-induced cardiac cell injury via expression changes of multiple genes plays a critical role in the pathogenesis of numerous heart diseases. MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate about 30% of the genes in a cell via degradation or translational inhibition of their target mRNAs. Currently, the effects of ROS on miRNA expression and the roles of miRNAs in ROS-mediated injury on cardiac myocytes are uncertain. Using quantitative real-time RT-PCR (qRT-PCR), we demonstrated that microRNA-21 (miR-21) was upregulated in cardiac myocytes after treatment with hydrogen peroxide (H2O2). To determine the potential roles of miRNAs in H2O2-mediated gene regulation and cellular injury, miR-21 expression was downregulated by miR-21 inhibitor and upregulated by pre-miR-21. H2O2-induced cardiac cell death and apoptosis were increased by miR-21 inhibitor and was decreased by pre-miR-21. Programmed cell death 4 (PDCD4) that was regulated by miR-21 and was a direct target of miR-21 in cardiac myocytes. Pre-miR-21-mediated protective effect on cardiac myocyte injury was inhibited in H2O2-treated cardiac cells via adenovirus-mediated overexpression of PDCD4 without miR-21 binding site. Moreover, Activator protein 1 (AP-1) was a downstream signaling molecule of PDCD4 that was involved in miR-21-mediated effect on cardiac myocytes. The results suggest that miR-21 is sensitive to H2O2 stimulation. miR-21 participates in H2O2-mediated gene regulation and functional modulation in cardiac myocytes. miR-21 might play an essential role in heart diseases related to ROS such as cardiac hypertrophy, heart failure, myocardial infarction, and myocardial ischemia/reperfusion injury.

MicroRNA-21 targets tumor suppressor genes in invasion and metastasis

MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that target protein-coding mRNAs at the post-transcriptional level. Our previous studies suggest that mir-21 functions as an oncogene and has a role in tumorigenesis, in part through regulation of the tumor suppressor gene tropomyosin 1 (TPM1). Given that TPM1 has been implicated in cell migration, in this study we further investigated the role of mir-21 in cell invasion and tumor metastasis. We found that suppression of mir-21 in metastatic breast cancer MDA-MB-231 cells significantly reduced invasion and lung metastasis. Consistent with this, ectopic expression of TPM1 remarkably reduced cell invasion. Furthermore, we identified two additional direct mir-21 targets, programmed cell death 4 (PDCD4) and maspin, both of which have been implicated in invasion and metastasis. Like TPM1, PDCD4 and maspin also reduced invasiveness of MDA-MB-231 cells. Finally, the expression of PDCD4 and maspin inversely correlated with mir-21 expression in human breast tumor specimens, indicating the potential regulation of PDCD4 and maspin by mir-21 in these tumors. Taken together, the results suggest that, as an oncogenic miRNA, mir-21 has a role not only in tumor growth but also in invasion and tumor metastasis by targeting multiple tumor/metastasis suppressor genes. Therefore, suppression of mir-21 may provide a novel approach for the treatment of advanced cancers.

MicroRNA Control of Hematopoietic Stem Cell Fitness and Myelopoiesis.

The Growth factor independent -1 (Gfi1) transcriptional repressor regulates both hematopoietic stem cell (HSC) self renewal and myeloid differentiation. Gfi1 null mice eventually die of HSC exhaustion, and Gfi1−/− HSC are not competitive in transplantation assays. Here we show that Gfi1 is a master regulator of microRNAs and that transcriptional control of a microRNA gene is critical for Gfi1-directed stem cell competitiveness and myelopoiesis. First, we show that the gene encoding miR21 is a direct transcriptional target of Gfi1. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal Gfi1 binding to specific DNA sequences upstream of the miR21 stem loop. Moreover, the expression of Gfi1 and miR21 is reciprocal in 1) wild type and Gfi1−/− marrow cells, 2) during normal differentiation from common myeloid progenitors (CMP) to granulocyte monocyte progenitors (GMP), and 3) during treatment-induced differentiation of human myeloid leukemia cell lines. Forced expression of Gfi1 lowers miR21 levels in wild type Lin− bone marrow cells and human cancer cell lines. Knockdown of Gfi1 expression with shRNA in human cancer cell lines increases miR21 expression. Moreover, conditional deletion of Gfi1 induces miR21 expression in primary murine hematopoietic cells, including sorted CMP and GMP. Thus, Gfi1 transcriptionally regulates miR21 in both human and murine hematopoietic cells. Interestingly, we find that the Ski oncoprotein/transcriptional corepressor is a direct target of miR-21. Subsequently, Ski is dramatically reduced in Gfi1−/− HSC and in wild type bone marrow Lin− cells forced to express miR21. Gfi1 may repress miR21 to maintain functional competence. Specifically, we find that Ski is a previously undescribed Gfi1 corepressor. Both endogenous Ski and Gfi1 physically interact. Synthetic Ski and Gfi1 proteins reveal that the interaction is both strong and specific. Chromatin immunoprecipitation reveals Ski and Gfi1 occupy several Gfi1 target genes. However, Ski function is critical as a corepressor on only a subset of Gfi1 target genes. To determine the importance of Ski corepression to Gfi1 induced biology, we examined two well established phenotypes of Gfi1 loss of function; HSC competitiveness and myelopoiesis. When Gfi1−/− embryonic stem cells are injected into a wild type blastocyst, they do not participate in hematopoiesis. Similarly, we find that when Ski−/− embryonic stem cells are injected into a blastocyst, they infrequently participate in hematopoeisis. Next, because Ski−/− animals die at or before birth, we examined the fitness of Ski−/− fetal liver HSC. In competitive transplantation assays, Ski−/− fetal liver HSC were significantly impaired in reconstitution compared to congenic wild-type competitor fetal-liver HSC. Moreover, Ski null HSC generated significantly less myeloid progeny. Thus, Ski−/− HSC display a partial phenocopy of Gfi1−/− hematopoiesis. We conclude that Gfi1 directly targets miR21 to control the expression of Ski, a corepressor for Gfi1, and that the Gfi1/Ski complex is critical to regulate a subset of Gfi1 target genes important for HSC fitness and myeloid cell production.

MicroRNA-21 Regulates Expression of the PTEN Tumor Suppressor Gene in Human Hepatocellular Cancer

microRNAs (miRNAs) are short noncoding RNAs that regulate gene expression negatively. Although a role for aberrant miRNA expression in cancer has been postulated, the pathophysiologic role and relevance of aberrantly expressed miRNA to tumor biology has not been established. We evaluated the expression of miRNA in human hepatocellular cancer (HCC) by expression profiling, and defined a target gene and biologically functional effect of an up-regulated miRNA. miR-21 was noted to be highly overexpressed in HCC tumors and cell lines in expression profiling studies using a miRNA microarray. Inhibition of miR-21 in cultured HCC cells increased expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, and decreased tumor cell proliferation, migration, and invasion. In contrast-enhanced miR-21 expression by transfection with precursor miR-21 increased tumor cell proliferation, migration, and invasion. Moreover, an increase in cell migration was observed in normal human hepatocytes transfected with precursor miR-21. PTEN was shown to be a direct target of miR-21, and to contribute to miR-21 effects on cell invasion. Modulation of miR-21 altered focal adhesion kinase phosphorylation and expression of matrix metalloproteases 2 and 9, both downstream mediators of PTEN involved in cell migration and invasion. Aberrant expression of miR-21 can contribute to HCC growth and spread by modulating PTEN expression and PTEN-dependent pathways involved in mediating phenotypic characteristics of cancer cells such as cell growth, migration, and invasion.